The recordation of images, especially fluorescence images, in microscopy takes place today often with digital cameras such as CCD cameras. For this purpose, cameras today are used in different configurations and with different recording quality. In the selection of a camera, the parameters of recording time and resolution are, as a rule, mutually opposed. For a high image rate, only a limited recording quality can be achieved. High resolution digital cameras with correspondingly higher spatial resolution require an adequate exposure time for the reduction of the image noise per image spot. Further differences lie in the type of the color recording with or without color dividers and color filters in the light path and therefore sequential or parallel recording of the color channels.
In the parallel recording of color images with three color channels of a one-chip sensor, the basic problem is present that the actually available resolution of the sensor is at least halved by the color mask which is introduced. Here, for example, a green mask is disposed on the one half of the light-sensitive elements (pixels) and the other half is subdivided between red and blue with equal parts. Correspondingly, the available spatial resolution becomes less in the respective spectral channels. In cameras of this kind, as a rule, a mathematical interpolation between the measured image points is used in order to generate a color image having the basic resolution of the CCD sensor.
German patent 3,837,063 discloses a method known as the so-called “color-co-site-sampling method”. With this method, the resolution loss caused by the color mask is completely compensated. For this purpose, the CCD sensor is displaced by a highly precise piezo-mechanical device relative to the image to be recorded in such a manner that complete partial images are recorded with the three color channels (R-G-B) sequentially at respectively different positions. Thereafter, and without interpolation, a complete image is developed by subsequent interlacing of the sequentially recorded partial images. The displacement takes place in dependence upon the color mask so that one and the same point in the object is detected sequentially by the three color channels (for example, 2x green, 1x red and 1x blue).
The scanning of an image in both directions can be increased up to a factor of three via an additional microscanning. Correspondingly, the number of recorded image points can be increased up to a further factor of nine; a further increase of the scanning is not purposeful because of the finite aperture.
The microscanning technique for increasing the resolution, however, causes that an image P is compiled from several sequentially recorded partial images Pn. For this reason, the method is sensitive to movements of the object or changes in luminosity. During recording of fluorescent images, the stability and brightness of the object is a problem. Because of the illumination exciting the fluorescence, the specimen undergoes a bleaching, a so-called photobleaching, and thereby a reduction of the intensity of the fluorescence, a so-called fading. With the compilation of the sequentially recorded partial images, artefacts then occur in the complete image, which are caused by the different fluorescence intensities of the sequentially recorded partial images.
To eliminate fading artefacts in the compilation of sequentially recorded partial images, U.S. Pat. No. 5,682,567 discloses compensating the intensity of the fluorescence, which becomes less from one image to the next, by a corresponding lengthening of the recording time. The correction of the exposure time required for this is determined in each case in advance of the data acquisition. The artefacts in the compiled image are, however, only avoided if the fluorescence intensities actually follow the law which forms the basis of the computation of the corrected exposure times.